run in TBE £0.5 at room temperature for 2 h at 150 V. The following two (Q/q) 27-bp unmethylated oligonucleotides were used: 5 0 -GATCCTTCGCCTAGGCTC(A/G)CAGCG CGGGAGCGA-3 0 . A methylated q probe (q*) was generated by incorporating a methylated cytosine at the mutated CpG site during oligonucleotide synthesis. Transient transfection assayThe constructs contained 578 bp from IGF2 intron 3 (nucleotides 2868-3446), followed by the IGF2 P3 promoter (nucleotides 2222 to þ45 relative to the start of transcription) 12 and a luciferase reporter. C2C12 myoblast cells were grown to approximately 80% confluence. Cells were transiently co-transfected with the firefly luciferase reporter construct (4 mg) and a Renilla luciferase control vector (phRG-TK, Promega; 80 ng) using 10 mg Lipofectamine 2000 (Invitrogen). Cells were incubated for 25 h before lysis in 100 ml Triton lysis solution. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The results are based on four triplicate experiments using two independent plasmid preparations for each construct. Statistical analysis was done with an analysis of variance.

Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter-enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

Regulation of patterning and morphogenesis during embryonic development depends on tissue-specific signaling by retinoic acid (RA), the active form of Vitamin A (retinol). The first enzymatic step in RA synthesis, the oxidation of retinol to retinal, is thought to be carried out by the ubiquitous or overlapping activities of redundant alcohol dehydrogenases. The second oxidation step, the conversion of retinal to RA, is performed by retinaldehyde dehydrogenases. Thus, the specific spatiotemporal distribution of retinoid synthesis is believed to be controlled exclusively at the level of the second oxidation reaction. In an N-ethyl-N-nitrosourea (ENU)-induced forward genetic screen we discovered a new midgestation lethal mouse mutant, called trex, which displays craniofacial, limb, and organ abnormalities. The trex phenotype is caused by a mutation in the short-chain dehydrogenase/reductase, RDH10. Using protein modeling, enzymatic assays, and mutant embryos, we determined that RDH10 trex mutant protein lacks the ability to oxidize retinol to retinal, resulting in insufficient RA signaling. Thus, we show that the first oxidative step of Vitamin A metabolism, which is catalyzed in large part by the retinol dehydrogenase RDH10, is critical for the spatiotemporal synthesis of RA. Furthermore, these results identify a new nodal point in RA metabolism during embryogenesis.[Keywords: Retinoic acid; Vitamin A; orofacial cleft; limb] Supplemental material is available at http://www.genesdev.org.

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTENdeficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector β-catenin. Akt phosphorylates β-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of β-catenin. Accession codes. Gene Expression Omnibus (GEO): GSE6078.URLs. GEO: http://www.ncbi.nlm.nih.gov/projects/geo/.Supplementary information is available on the Nature Genetics website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. HHS Public AccessAuthor manuscript Nat Genet. Author manuscript; available in PMC 2015 December 16. Published in final edited form as:Nat Genet. 2007 February ; 39(2): 189-198. doi:10.1038/ng1928. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe failure of most current therapies to cure cancer has led to the hypothesis that treatments targeted at malignant proliferation spare a more slowly cycling 'cancer stem cell' population that has the ability to regenerate the tumor 1 . Recently, cancer stem cells have been identified and shown to seed tumors upon transplantation into a secondary host [2][3][4] . However, little is known about the process by which mutation(s) in a stem cell result in primary tumor initiation.Although there are many 'causes' of intestinal cancer, it is well established that almost all cases begin with the development of benign polyps, mainly involving benign neoplastic proliferation of epithelium. The epithelium of the small intestine is composed of a proliferation compartment (crypt) and a differentiation compartment in the villus (Fig. 1a). ISCs, located near the crypt base and above Paneth cells 5,6 , give rise to enterocytes, goblet cells, enteroendocrine cells and Paneth cells [6][7][8] . Intestinal polyposis features a substantial increase in the number of crypts (crypt expansion) and a reduction in epithelial cell differentiation 6,7,9,10 . A key question 7,9,11 is whether stem cells are involved in the abnormal crypt expansion during polyp initiation.Studies of human hereditary intestinal polyposis syndromes (which typically, but not uniformly, predispose affected individuals to gastrointes...

Epithelial stem cells (EP-SCs